Affinity assays are analytical methods based on non-covalent affinity binding. Among them are immune tests or resp. immunoassays which happen to be standard operations in clinical chemistry, diagnostics, environmental analytics and bio-process control. The basis of any affinity assay is at least one specific perception reaction, for instance the specific binding of an affinity ligand to an antibody. Instead of immune-proteins, also other affinity receptors like lectines or aptamers, can be used for selective binding to an analyte.
Inherent specificity and high affinity of affinity binding enables selective, highly specific and highly sensitive concentration measurement. By means of antibodies, it is possible to record picomolar or nanomolar solutions of numerous analytes. Specific antibodies can be produced for almost all kinds of chemical compounds, from low-molecular weight substances, so-called haptenes, up to biological or synthetic macromolecules. Methods of establishing and implementation of affinity assays are well known in professional circles and can be read in a large number of publications (by example James P. Gosling “Immunoassays”, Practical Approach Series, Oxford University Press, Oxford, UK).
An immunoassay or affinity assay normally consists of several steps, a.o. at least one incubation step, at least one washing step and a concentration measurement.
Modern techniques have been developed over the last years, which simplify the handling of immunoassays and thus making them accessible to new applications such as the pregnancy test in a control strip format (immunochromatography). Another direction of development is to incorporate a multitude of immune tests on a small area, the “Biochip” or “Microarray”. Some immune tests in microarray format have the ability to detect a multitude of analytes from a single blood sample. One of the problems related to miniaturization of immunoassays consists in decrease of signal strength by reduction of reagent amount.
Since the common affinity receptors (e.g. antibodies and antigens) and their binding partners are not directly detectable in a sensitive way by usual detectors, they are chemically linked to a labeling substance (“label” or “marker”) providing a detectable signal depending on analyte concentration. For miniaturized immunoassays, labels of the most specific activity are required, e.g. enzymes (a. o. peroxidase, alkalic phosphatase) or fluorescent dyes with high fluorescence recovery rates. Using enzymes, an amplification is achieved by catalyzing the generation of a signal substance (e.g. a colored reaction product). Fluorescent dyes allow the fastest and most direct detection, however, high sensitivity of fluorescence affinity assays requires extensive high-performance optical devices.
High-molecular weight antigens such as proteins can be detected in a particularly favorable way by a “sandwich”-immunoassay. In its most common implementation, the surface is coated by a specific antibody so that, after incubation with a sample solution, the analyte is linked to the surface, whereas other components of the sample are removed during a subsequent washing procedure. The actual detection takes place after incubation with a second, labeled specific antibody and yet another washing procedure (and, if necessary, a chromogenic reaction when using enzyme labels). The dose-effect correlation is positive.
The quantitative ascertainment of substances with a molecular weight below 1000 Dalton is one of the most important application fields of immunoassays. For these purposes, almost exclusively so-called competitive assays are applied. Here, the non-labeled analyte competes against an analog for the free binding places of an affinity receptor. The latter or the competing analog, which can be a high- or low-molecular weight soluble substance, carry the label.
In competitive affinity assays for the detection of low-molecular weight analytes, normally the labeled molecules are split into a soluble and a surface-bound fraction whereas the proportion of these fractions is dependent on the analyte concentration. Affinity binding of the labeled molecules to a surface coated with immobilized reactants takes place in many cases. The amount of surface-bound label is measured after flushing away the soluble fraction. In the absence of the competing analyte, the maximum amount of labeled molecules can be bound to the surface; the amount of surface-bound labeled molecules is reduced by the specific binding of the analyte to its receptor.
Since the amount of bound labeled substance is measured after a washing procedure, an inverse dose-effect correlation results, i.e. the strongest signals are attained at the lowest analyte concentrations; at very high analyte concentrations a low dependence of signal strength on concentration occurs, at very low analyte concentrations the uncertainty of the differential measurement is interfering. Due to the principle of difference measurement, the concentration of the labeled reactant has to be adjusted to that of the analyte. As the immobilized amount of labeled substance normally forms a very thin surface layer, detector performance requirements are high for small measuring areas, what has an impact on expenses.
It would be desirable either to overcome the inverse characteristics of competitive assays or to achieve a ratiometric measurement of the fraction of labeled molecules formed by the reaction with the analyte. The heterogeneous test system has the additional disadvantage of unavoidable unspecific binding of labeled molecules to the surface, thus causing more or less noise. It would be of great advantage, if competitive affinity assays could be carried out completely in homogeneous phase.
The mentioned limitations of competitive immunoassays mostly rely on the fact that only the surface-bound fraction of the labeled binding partner (e.g. of the antibody or the antigen) is being detected. At present, there is a strong requirement for miniaturized immunoassays with positive dose-effect correlation and/or without the necessity of linking the labeled molecules to a surface.